1. Field of the Invention
The present invention relates generally to the fields of radiation biology and cell biology. More particularly, it concerns the attenuation of the effect of ionizing radiation induced activation of cytokines, such as tumor necrosis factor, by inhibitors of extranuclear signal transduction.
2. Description of the Related Art
Tumor necrosis factor (TNF) is a polypeptide mediator of the inflammatory response and induces proliferation of fibroblasts, recruitment of inflammatory cells, activation of endothelial cells and hemorrhagic necrosis of tumors in mice (Fiers, 1991). Tumor necrosis factor also kills tumor cells directly through the induction of free radical formation and DNA fragmentation. These effects may explain the mechanism by which TNF enhances tumor cell killing by x-rays (Hallahan et al., 1990, Hallahan et al., 1989). Increased production of TNF is associated with inflammatory disorders such as autoimmune demyelination of the central nervous system and respiratory distress syndrome (Fiers, 1991).
Tumor necrosis factor is produced by monocytes and macrophages in response to diverse stimuli, including ionizing radiation (Sherman et al., 1991, Wasserman et al., 1991). Increased TNF serum levels are associated with acute and subacute sequelae following total body irradiation (Holler et al., 1990). Radiation sequelae that correlate with TNF serum levels include pneumonitis, endothelial leakage syndrome, and veno-occlusive disease. Although, the effects of ionizing radiation on rapidly proliferating cell renewal systems have classically been theorized to be due to direct killing of stem cells within the injured organ, other work has suggested that TNF induction plays a role in the acute effects of irradiation by directly enhancing cell killing as well as mimicking the acute inflammatory response (Wong et al., 1991).
Signaling pathways activated by DNA damage contribute to survival of prokaryotes and eukaryotic cells following exposure to x-rays or UV light. In irradiated E. coli, damaged DNA forms a complex with the Rec A protease resulting in the transcriptional induction of a variety of genes including those encoding DNA repair enzymes (Walker, 1985). In yeast, UV light and x-rays result in the induction of genes which participate in the repair of damaged DNA (Jones et al., 1991, Cole et al., 1987). Genes whose products are proposed to recognize damaged or un-replicated DNA and to participate in intracellular signaling that regulates cell cycle progression and DNA repair have been identified in S. cerevisiae and S. pombe (House et al., 1992, Enoch et al., 1992). The complexity of this signaling pathway is demonstrated by the number of genes involved in sensing DNA damage and transmitting the signal (Enoch et al., 1992). DNA damage is presumed to be the initiating event in mammalian cell induction of stress response genes following x-ray or UV exposure (Herrlich et al., 1992, Kastan et al., 1992). However, the mechanisms of DNA damage recognition have not been identified in mammalian cells.
Signal transduction pathways activated by ionizing radiation include increased phosphotransferase activity of cytoplasmic protein kinases (Hallahan et al., 1991a, Hallahan et al., 1991b, Uckun et al., 1992). Moreover, inhibition of protein kinases blocks radiation-mediated gene induction and effects diverse biological endpoints such as apoptosis (Uckun et al., 1992), radiation survival (Hallahan et al., 1992)) and induction of the cytokine tumor necrosis factor (TNF) (Hallahan et al., 1991b). The calcium/phospholipid-dependent protein kinase (PKC) is activated within 15 seconds of ionizing radiation exposure and is extinguished by 90 seconds in human leukemia HL-60 cells (Hallahan et al., 1991b).
Second messengers that participate in PKC activation following exposure to external stimuli include free fatty acids such as arachidonic acid (Nishizuka, 1992). Previous studies have suggested that phospholipase A2-mediated hydrolysis of oxidized membrane phospholipids is a primary means of bioreduction following oxidative injury (Au et al., 1983, Sevanian et al., 1983)(reviewed in van Kuijk et al., 1987). For example, arachidonic acid release is increased following treatment with H.sub.2 O.sub.2 due to hydrolysis of oxidized membrane phospholipids (Gustafson et al., 1991, Shasby et al., 1988). To determine whether arachidonic acid production was associated with radiation-mediated signal transduction, arachidonic acid production was quantified in irradiated HL-60 cells and found an increase in arachidonate within 30 minutes following irradiation.
Since phospholipase A2 hydrolysis phosphatidylcholine to arachidonic acid, the effects of the phospholipase A2 inhibitors mepacrine (Rao et al., 1993), and bromphenylbromide (BPB) (Peppelenbosch et al., 1993) were investigated. In addition, the effects of dexamethasone and pentoxifylline on radiation-induced fatty acid hydrolysis were studied, as these agents have been shown to inhibit phospholipase A2, reduce the production of cellular mediators of inflammation and tissue injury, and inhibit lipopolysaccharide-induced TNF production in monocytes (Strieter et al., 1988, Hah et al., 1990). Moreover, glucocorticoids and pentoxifylline are employed clinically to prevent some acute toxicities of radiotherapy (Bianco et al., 1991, Phillips et al., 1975). The inventors determined that each agent attenuated arachidonic acid release into the medium of cells treated with X-rays or H.sub.2 O.sub.2. Thus, extranuclear second messengers are in part responsible for radiation-mediated signal transduction and inhibition of this pathway may provide a means of attenuating the inflammatory-like response observed in irradiated tissues through the inhibition of TNF gene induction.
Radiation-mediated TNF induction is attenuated by inhibitors of phospholipase A2 and protein kinase C (PKC) Hallahan et al., 1994 (in press), Hallahan et al., 1991b). Phospholipase A2 hydrolyzes the membrane fatty acid phosphatidylcholine to form arachidonic acid and eliminates oxidized membrane lipid following exposure to oxidizing agents (Sevanian et al., 1991, van Kuijk et al., 1987). Inhibitors of this enzyme attenuate arachidonate production and PKC activation following treatment with X rays or H.sub.2 O.sub.2 (8). Arachidonic acid is subsequently metabolized by lipoxygenase or cyclooxygenase to form leukotrienes or prostaglandins respectively. Moreover, arachidonic acid has been shown to activate PKC (Fan et al., 1990). Protein kinase C is rapidly and transiently activated following irradiation and mediates radiation-induction of certain genes, including TNF, Egr-1 and c-jun (Hallahan et al., 1991a, Hallahan et al., 1991b). Egr-1 and c-jun encode transcription factors and are associated with G1/S transition following mitogenic stimulation (Sukhatme et al. 1990). Since these radiation inducible genes are known to be induced through activation of both PKC-dependent and -independent pathways, the inventors investigated whether these signal transduction pathways are specific for radiation-mediated TNF induction.
Phospholipase A2 inhibitors used in clinical radiotherapy to ameliorate acute and subacute sequelae include glucocorticoids and pentoxifylline (Bianco et al., 1991, Phillips et al., 1975). Glucocorticoids are used to treat radiation induced proctitis, pneumonitis, conjunctivitis, external otitis, CNS syndromes and occasionally mucositis. Pentoxifylline is effective in preventing pneumonitis and mucositis following total body irradiation prior to bone marrow transplantation (Bianco et al., 1991). Taken together, these findings implicate phospholipase A2 in radiation-induced TNF induction and the acute sequelae of radiotherapy. Because arachidonic acid is metabolized by lipoxygenase and cyclooxygenase, inhibitors of these enzymes were added to cells prior to irradiation to determine whether signaling through these pathways contribute to radiation-induction of TNF and related molecules.
A surprising and unexpected advantage of the instant invention is the ability to selectively inhibit the expression of TNF or genes coupled to the TNF promoter, while simultaneously allowing expression of genes operatively linked to, for example, other radiation inducible promoters such as Egr and jun. This allows the selective induction of genes linked to radiation inducible promoters without the corresponding increase in tumor necrosis factor that may be involved in clinical symptoms of acute and subacute radiation induced sequelae.